This invention relates to a process for the isolation of endosymbiont nitrogen-fixing bacteria and phosphorous-providing fungi in the roots of plants, and more particularly to an improved surface sterilization process for such roots to isolate the bacteria and fungi.
Nitrogen and phosphorus are essential mineral nutrients for plants. A shortage or lack of either nutrient in soils will adversely affect plant growth. Heretofore, nitrogen and phosphorus have been supplied to crops and other bromass producing plants by the addition of fertilizers to the soil. However, increasing costs of producing such fertilizers have led to searches for alternative methods of supplying these mineral nutrients to growing plants.
It is known that nitrogen-fixing root nodules occur on both leguminous and non-leguminous plants. Members of the legume family whose roots are infected by the soil bacterium Rhizobium are the primary source of nitrogen fixation in agricultural systems. Some non-leguminous angiosperms form root nodules when invaded by soil actimomycetes bacteria which also enable fixation of atmosphere nitrogen by these plants. Additionally, it is known that certan fungi produce ectomycorrhizal and endomycorrhizal growths on plant root systems which can provide phosphorus to several plant species including some which are agriculturally important.
Presently, the successful growth of nitrogen-fixing bacteria or phosphorus-providing fungi on a particular host plant depends on the presence of an endogenous population of such bacteria or fungi in the soil in which the plant is growing. When planted in soils having a low concentration of mineral nutrients, the absence of early and effective growth of root nodules and/or mycorrhiza delays the establishment and growth of seedlings and may even result in their complete failure. Previous methods of inoculating seedlings with suspensions of ground-up nodules or applying soil suspensions taken from where host plants were growing gave unpredictable and variable results because such suspensions contained a wide range of soil microorganisms as well as complex products derived from broken plant tissues.
Clearly, the need exists for techniques which will enable the isolation and cultivation of the causative bacteria and fungi which can then be used to inoculate seeds and/or seedlings. While isolation and identification of Rhizobium bacteria from legume root nodules have been known for sometime, only recently have successful isolation techniques been developed for certain non-leguminous plants. See, Callaham et al., Science, volume 199, pp. 899-902 (1978). Such techniques include surface sterilization of root nodules using sodium hypochlorite or mercury chloride solutions followed by microdissection and enzymetic digestion or suspension and dilution of the host tissues. Other reported techniques for isolating such bacteria from root nodules include Sephadex and sucrose density fractionation.
However, these techniques have not proved to be entirely satisfactory. Not only are they time consuming and technically complex, they exhibit a very high failure rate in successfully isolating the causative bacteria. Presently utilized surface sterilization techniques, designed to kill the wide variety of other soil microorganisms found on root nodules while leaving unaffected the causative bacteria and fungi, have not been successful. This is due to several factors. One factor is that air pockets and small pathways in the interior of such root nodules harbor a variety of microorganisms which the surface sterilant does not reach. When the root nodules are crushed or dissected, these microorganisms are transferred along with the desired endophytes to a cultivation medium, causing contamination of the medium. Another factor is that the surface sterilants heretofore used in many cases act so quickly on the root nodules and mycorrhizal growths that all of the desired microorganisms are killed along with the rest of the microorganisms.
Accordingly, the need exists in the art for an improved isolation technique for nitrogen-fixing bacteria and phosphorus-providing fungi which is both simple to perform and results in a high success rate in providing an isolated culture.